Mutation of both alleles of the RB1 gene (M1, M2) initiates the development of retinoblastoma. We will define the genes in which additional mutations (M3-Mn) are required for progression of RB1 -/- cells to retinoblastoma. Future strategies to counter M3-Mn mutations will reduce the number of tumors in RB1 +/- predisposed persons and lead to novel therapies. Only humans spontaneously develop retinoblastoma, arising in an undefined cell, not accessible to experimental manipulation. An excellent model of retinoblastoma (TAg-RB) is induced by expression of the SV40 viral oncogene, TAg, in murine retina. Although TAg inactivates many proteins, it is still insufficient for development of retinoblastoma without further M3-Mn events. We hypothesize that: Some of the M3-Mn events that contribute to development of retinoblastoma will be common to spontaneous human and induced murine retinoblastoma and involve disruption of apoptosis. Retinoblastoma will be induced in vitro in retinal explants and in vivo in genetically modified mice when RB family proteins (MI/M2) are inactivated and M3-Mn mutational events have occurred. Definition of the common additional M3-Mn events downstream of mutation of RB1 will provide target(s) to inhibit retinoblastoma development and/or progression. Specific Aim 1: To identify and compare the spectrum of M3-Mn genes in human retinoblastoma and TAg-RB by comparing regions of genomic gains and losses (murine aCGH compared to human CGH) and expression profiles (general, cell cycle- and retina- specific microarrays). Specific Aim 2: To evaluate the roles of three candidate M3-Mn genes (decrease in p75 NTR, overexpression of RBKIN and loss of CDH11), in human and TAg-RB retinoblastoma. Specific Aim 3: To functionally assess the roles of candidate M3-Mn genes in vitro by blocking expression of candidate tsgenes and overexpressing candidate oncogenes in murine retinal explants that already have MI/M2 (RB1 -/- knockout or TAg-RB). Specific Aim 4: To functionally assess the roles of candidate M3-Mn genes in vivo by examining TAg- RB tumor incidence and progression in knockout M3-Mn mice crossed with TAg-RB mice.